Antimicrobial activity and safety features assessment of Weissella spp. from environmental sources

Abstract Weissella strains have been reported to be useful in biotechnological and probiotic determinations, and some of them are considered opportunistic pathogens. Given the widespread interest about antimicrobial susceptibilities, transmission of resistances, and virulence factors, there is little research available on such topics for Weissella. The aim of this study was to assess the safety aspects and antimicrobial potential of 54 Weissella spp. strains from different environmental sources. Antibiotic susceptibility, hemolytic activity, horizontal transfer, and antibacterial activity were studied, as well as the detection of biogenic amine BA production on decarboxylase medium and PCR was performed. All the strains were nonhemolytic and sensitive to chloramphenicol and ampicillin. Several strains were classified as resistant to fusidic acid, and very low resistance rates were detected to ciprofloxacin, tetracycline, streptomycin, lincomycin, erythromycin, and rifampicin, although all strains had intrinsic resistance to vancomycin, nalidixic acid, kanamycin, and teicoplanin. Two BA‐producing strains (W. halotolerans FAS30 and FAS29) exhibited tyrosine decarboxylase activity, and just one W. confusa FS077 produced both tyramine and histamine, and their genetic determinants were identified. Ornithine decarboxylase/odc gene was found in 16 of the Weissella strains, although 3 of them synthesize putrescine. Interestingly, eight strains with good properties displayed antibacterial activity. Conjugation frequencies of erythromycin from Bacillus to Weissella spp. varied in the average of 3 × 10−9 transconjugants/recipient. However, no tetracycline‐resistant transconjugant was obtained with Enterococcus faecalis JH2‐2 as recipient. The obtained results support the safe status of Weissella strains, derived from environmental sources, when used as probiotics in animal feed.


| INTRODUC TI ON
The genus Weissella includes Gram-positive heterofermentative lactic acid bacteria LAB, asporogenous short bacilli, or coccoid bacilli that can be found in pairs or short chains. Phylogenetically, bacteria within this genus belonging to the Leuconostocaceae family were previously grouped along with the Leuconostoc and Lactobacillus (Björkroth et al., 2009;Collins et al., 1993). Currently, 24 distinct species of Weissella were validated Heo et al.2019;Hyun et al., 2021;Li et al., 2020;Lin et al., 2020;Praet et al., 2015). Weissella spp. are broadly disturbed in a range of ecological niches where they are hypothesized to have a probiotic effect , such as plants, vegetables, soil, water, and fermented foods of both plant or animal origin, as well as in feces, breast milk, animal skin and milk, and mucous membranes of humans and animals (oral, gastrointestinal tract, and vagina). Despite the fact that Weissella is a fairly recent genus in comparison to other LAB, it has been the subject of many studies during the past few years and has attracted the interest for use in the pharmaceutical, food, and medical sectors. It has been shown that some Weissella spp., specially W. confusa and W. cibaria, are able to produce exopolysaccharides EPS, mainly dextran, as natural food thickeners, and nondigestible oligosaccharides, or as prebiotics. These polymers make it of high interest for the development of applications combining EPS technological and nutritional benefits, predominantly for bakeries and the production of functional beverages (Baruah & Goyal, 2015;Juvonen et al., 2015;Korcz & Varga, 2021;Patel et al., 2012). Furthermore, the antimicrobial activity of several Weissella spp. has been observed against a wide range of pathogens via secondary compound production, and their potential use as probiotics has been investigated (Fhoula et al., 2018;Fusco et al., 2015;Kariyawasam et al., 2019;Trias &Bañeras, 2008;Yu et al., 2019). In relation to the healthpromoting benefits of putative probiotic Weissella, soe strains, primordially those belonging to W. cibaria, have been shown to have antiviral, immune-modulating, antiobesity, anticancer, anticholesterol, and antioxidant properties (Oh & Lee, 2021;Kang et al., 2011;Park et al., 2012;Kwak et al., 2014;Fhoula et al., 2018, andYu et al., 2019).
Despite these characteristics, the utilization of Weissella spp. as commercial starters or probiotics has not yet been explored. Until now, Weissella spp. are not generally recognized as safe (GRAS) nor as qualified presumption of safety (QPS) (Fessard & Remize, 2017). Kang et al. (2019) reported that two W. cibaria (CMU and CMS1) are commercially available as oral care probiotics in Korea, and registered as safe raw materials by the Korea Food and Drug Administration, although they have not yet been determined to be GRAS. This missing can be explained in part by the antibiotic resistance profile, biogenic amine synthesis, or infection risk (Fessard & Remize, 2017). In fact, scientists are opposed on whether or not to use Weissella spp., which are generally categorized as opportunistic pathogens, and occasionally linked with illnesses in people, who had weakened immune system (Fairfax et al., 2014;Fessard & Remize, 2017;Kamboj et al., 2015;Kumar et al., 2011;. More investigation into the safety of these strains' usage as probiotics in feed/food is required. Weissella spp. would have to get the safety proof to obtain GRAS accreditation through safety investigations (Fessard & Remize, 2017). Controversially, Weissella strains are still being used in the food and pharmaceutical industries, according to a vast number of scientific investigations (Teixeira, da Silva, et al., 2021).
This study aimed to evaluate the safety and determine the antibacterial activity of 54 Weissella spp. strains from distinct environmental sources in order to identify novel probiotic in foods or animal feeds. It could be used as an alternative to antibiotics, and to improve our knowledge about its safety and probiotic properties that may lead to its future use. To check in Weissella strains, the antibiotic resistance patterns, toxic compounds production, and any harmful genetic traits that may be transferred to other bacteria contributed to the selection of potential safety strains from novel origin.  (Fhoula et al., 2013(Fhoula et al., , 2018Fhoula et al., 2022 (unpublished work)

| Hemolytic activity
Hemolytic activity was determined by streaking bacterial cultures on Columbia agar plates supplemented with 5% of horse blood (bioMérieux) and then hemolysis zones around the colonies were checked (Maragkoudakis et al., 2006). All experiment was performed in three replicates.

| Antibacterial activity against pathogen and food-borne bacteria
The antibacterial activity was determined using the agar welldiffusion method described by Tagg and McGiven (1971). Seven indicator strains were used to assess the growth inhibition activity of tested. To remove the effects of organic acid and hydrogen peroxide, the supernatants were treated with catalase (300 IU/ml, 37°C, 1 h, Sigma Aldrich) and neutralized with 1 M NaOH. These catalasetreated cell-free neutralized supernatants were then examined for antimicrobial activities, including as those due to bacteriocins. All indicator strains were grown in BHI broth at 37°C. Trypticase soy agar plates were overlaid with 5 ml of soft agar (0.75%) containing 50 μl of freshly grown culture. The wells were made in agar and filled with 100 μl of CSF of tested strain. After incubation at 37°C for 18 h, the diameter of the inhibition zones was measured. All antibacterial tests were performed in triplicate.

| Detection of potential biogenic amine producer
The amino acid decarboxylase activity of Weissella strains was assessed in the decarboxylase agar medium, as described by Bover-Cid and Holzapfel (1999), containing 1% of the appropriate precursor amino acids l-tyrosine, l-histidine, and l-ornithine hydrochloride (Sigma). The tested strains were spotted on the decarboxylase agar medium and incubated anaerobically at 37°C for 72 h. The presence of a purple color in the medium around the colonies indicated a positive reaction; however, a yellow color indicated a negative reaction.

| DNA extraction
Genomic DNA extraction of Weissella strains was performed enzymatically using a kit InstaGene TM Matrix (BioRad) according to the manufacturer's instructions.

| Transfer of antibiotic resistance
The transferability of erythromycin resistance of the B. thuringiensis sv kurstaki strain (Sm R Ery r ), potential donor, was evaluated using three recipient strains (W. halotolerans V10, W. paramesenteroides FS45, and W. confusa FS53) that are sensitive to erythromycin but resistant to nalidixic acid. To assess the transferability of tetracycline resistance of the two W. confusa (FS44 and FS63) obtained from olive rhizosphere soil, E. faecalis JH2-2 (FUS r , RIF r , and TET s ), free from plasmids, was chosen as the recipient strain. The filter mating procedure was used to investigate antibiotic resistance transfer, as reported by Gevers et al. (2003). Briefly, donor and recipient cell cultures (V/V of 1 ml), at exponential growth, were mixed and deposited onto a sterile nitrocellulose membrane filter (0.45 µm pore size, Milli-pore, USA), and the filter was incubated on nonselective medium agar based on the ideal growth conditions of the recipient strain. The bacteria were rinsed off the filters and suitable dilutions were seeded onto donor-, recipient-, and TC-selective agar plates.
Three replicates of all matings were conducted.

| Antimicrobial-resistant profiles and genetic determinants
Tables 1 and 2 summarize the prevalence and antibiotic resistance phenotypes perceived among tested Weissella strains based on the disk diffusion method. We recorded a high prevalence of resistance to fusidic acid in 48.1% of Weissella strains while a low resistance rate was observed to ciprofloxacin 14.8%, tetracycline 11.1%, streptomycin (high-level resistance) 7.4%, lincomycin 7.4%, and rifampicin 7.4%. All the tested strains were susceptible to ampicillin, chloramphenicol, and erythromycin, while resistant to vancomycin, teicoplanin, nalidixic acid, kanamycin, and streptomycin (low-level resistance). Intermediate resistance to rifampicin was seen in seven strains (13%), for lincomycin and erythromycin in three strains (5.6%), and for chloramphenicol in two strains (3.7%  (2015) and Suhonen (2019) for Weissella.
To elucidate the mechanism responsible for the resistance phenotypes perceived, genes encoding those phenotypes were checked by PCR as described above (Table 2).
The chloramphenicol MIC values (32 mg/L) obtained for the two W. confusa (FS44 and V20) were higher than the recommended breakpoint value (4-12/16 mg/L). No cat gene encoding chloramphenicol acetyltransferase has been detected in these strains (

| Biogenic amine production of Weissella
The presence of BA-producing Weissella was qualitatively investigated by assessing color variations in the decarboxylase medium.

| Detection of genes encoding histidine, tyrosine, and ornithine decarboxylases
To examine the presence of genes hdc, tydc, and odc in the 54 Weissella strains, which could reveal or not the BA production ability in the decarboxylase medium, we performed PCR amplification investigation. The results showed that decarboxylase-related gene odc was determined in 16 strains (29.6%), of which only three strains expressed phenotypically putrescine production. On the other hand, BA gene tydc was proved in three initially tyramine-producing strains W. halotolerans (FAS30 and FAS29) and W. confusa FS77.
Besides, BA gene hdc was determined only in histamine-producing W. confusa FS77.

| Transferability of antibiotic resistance genes ARGs
The ability of donors to transmit antibiotic resistance to the recipients was tested by filter mating approach. As shown in Table 5
On the other hand, resistance to teicoplanin, one of the glycopeptides, was also revealed in the genome of some Weissellas due to the presence of the vanZ resistance gene as it is with W. confusa LBAE C39-2, W. cibaria KACC 11,862, and W. paramesenteroides ATCC 33,313 (Abriouel et al., 2015). However, in our study,  Our results revealed that the incidences of chloramphenicol, tetracycline, and erythromycin resistance in the six strains were very low using the disc diffusion method. These findings were in line with studies declaring Weissella to be commonly susceptible to tetracycline, erythromycin, chloramphenicol, and ampicillin (Abriouel et al., 2015;Jeong & Lee, 2015). Remarkably, we noted very high MIC values of potential acquired resistance to erythromycin, tetracycline, and chloramphenicol, wherein we can find strains fully resistant to one or more clinically relevant antibiotics.
The antimicrobial-resistant Weissella strains are belonging to W. halotolerans, W. soli, and W. confusa. In line with a recent study (Patrone et al., 2021), in W. cibaria strains we looked at, there was no indication of phenotypic antibiotic resistance. We noted that the EFSA Panel on Additives and Products or Substances used in Animal  Basbülbül et al., 2015;Abriouel et al., 2015). In the present study, the susceptibility level of the strains to tetracycline is species and strain dependent. The most common tetracycline resistance mechanism is mediated by the tet(K) gene, encoding tetracycline efflux pump responsible for removing antibiotics to the outside of the cell, and followed by tet(S) and tet(M) genes, coding for ri-  (Abriouel et al., 2015). In most cases, enterococci carry frequently the tet(M) gene (Aarestrup et al., 2000). The tet(S) gene was initially discovered in L. monocytogenes strain BM4210 on a  (Charpentier et al., 1993); however, it has been also reported on the chromosome of L. mesenteroides LbE16 strain (Flórez et al., 2016).
The cat and ermB genes were selected as they are the deeply studied and the most common spread resistance genes among LAB (Hummel et al., 2007;Thumu & Halami, 2012 & Tuncer, 2020). According to Abriouel et al. (2015), the lack of reports on the molecular identification of antibiotic resistance genes in Weissellas may be related to the significant variability in resistance genes.
The unsuccessful transfer of tetracycline resistance from W. This is the first report to involve the antibiotic resistance determinants transfer of pAW63 (ermB) from Bacillus thurigiensis, which is ubiquitous in the environment, and closely associated with the food-borne pathogen Bacillus cereus, potentially enterotoxigenic (Frederiksen et al., 2006).
These results led to suggest that Weissella is not a good vector to transfer antibiotic resistance genes, which can occur at a low frequency under laboratory conditions. It is a weak candidate to receive virulent determinants from closest gram-positive pathogens. Consequently, further study incorporating mating settings is needed to assess the potential of Weissella spp. strains to spread antibiotic resistance.
As β-hemolysis is linked to pathogenicity, in our investigation, tested Weissella strains did not exhibit hemolysis activity which is essential criteria for the selection of potential good strains.
Biogenic amines (BA) can be found in a variety of protein-rich foods and fermented foods, and eating foods with excessive levels of these amines can generate toxicological consequences and health concerns (Durak-Dados et al., 2020;Ruiz-Capillas & Herrero, 2019;Santos, 1996). A variety of factors affect BA production, including the raw materials used, processing conditions, and microbes (Barbieri et al., 2019;Santos, 1996). The BA production in food by lactic acid bacteria has attracted a great interest and become the subject of considerable research because of their putative role in food poisoning (Barbieri et al., 2019;Ruiz-Capillas & Herrero, 2019 (Jeong & Lee, 2015;Takebe et al., 2016). Our study indicates that bacteria's main ability to decarboxylate amino acids is linked to their ecological niche from whence it originated as fermented foods, as well as strain specificity and amino acid decarboxylase gene diversity (Barbieri et al., 2019;Benkerroum, 2016;Jeong & Lee, 2015). Generally, these findings led us to suggest that the strains from environment and animal sources do not produce biogenic amines. In line with Garai et al. (2007) and Jeong and Lee (2015), we could suggest that this capability is strain dependent rather than species specific. Interestingly, W. confusa F77, from camel feces, was able to inhibit the growth of Pa. larvae, the causative agent of American Foulbrood of honeybees, a notifiable bacterial disease that destroys larvae of honeybees in many countries (Ebeling et al., 2016). Then, F77 showed suitable properties that make it good for its use as a probiotic in the honeybee diet. LAB has been shown to be important in controlling this disease by several studies (Daisley et al., 2020;Lamei et al., 2020;Mudroňová et al., 2011).
Therefore, the selection and availability of Weissella with good functional characteristics (such as antibacterial activity, lack of phenotypic and genetic virulence determinants, and no horizontal gene transfer) make them more attractive for potential applications as probiotics or technological candidates in food, feed complement, and agriculture. More research is needed to increase our understanding of enzymatic activities, metabolic systems in Weissella spp., suggesting the potential use of these strains as novel probiotics to reduce infection and limit antibiotic utilization, such as the prevention of intestinal infections in cattle production (Patrone et al., 2021). To our knowledge, this is the first large-scale investigation detailing the antibacterial activity against numerous pathogens and the safety evaluation of Weissella spp. from diverse sources beyond the QPS procedure of LAB. This is one of the few publications describing the characterization and probiotic potential of Weissella spp. from original sources. In this study, the in vitro assessment was performed to investigate the antibacterial activity against pathogens, the antibiotic susceptibility, the lack of transferable antibiotic resistance determinants, and the prevalence of virulence factors, which resulted in the selection of eight strains (five W. confusa and three W. halotolerans). This approach is a useful strategy for preliminary large-scale selection of putatively safe Weissella strain for use as probiotics or supplements, as well as preventing the spread of bacterial cultures with harmful traits into the environment. Before the Weissellas can be considered recognizably safe probiotics, a full in vivo examination of their absence of cytotoxicity and undesirable effects must be carried out utilizing cell lines, raw food, and farm animals. Future investigations will be able to sustain the gained knowledge and assess the advantages.

ACK N OWLED G M ENTS
The authors acknowledge the financial support of the Tunisian Ministry of Higher Education and Scientific Research in the ambit of the laboratory project LR03ES03. The authors would like to thank Pr. Jacques Mahillon who offered B. thuringiensis for conjugation. The authors would also like to thank the assistance of Mrs. Mounira Msahli.

CO N FLI C T S O F I NTE R E S T
The authors declare that there is no conflict of interests.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M ED CO N S ENT
Written informed consent was obtained from all study participants.